Confirmation of direct interaction between H3K56cr and GLYR1 by H3K*56cr Site-Link in living cells
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Q Exactive HF
ORGANISM(S): Homo Sapiens (human)
TISSUE(S): Cell Culture, Early Embryonic Cell
SUBMITTER: 
Fangfei Qin
LAB HEAD: Peng R. Chen
PROVIDER: PXD033960 | Pride | 2023-03-06
REPOSITORIES: Pride
Publications
Linking chromatin acylation mark-defined proteome and genome in living cells.
Qin Fangfei F Li Boyuan B Wang Hui H Ma Sihui S Li Jiaofeng J Liu Shanglin S Kong Linghao L Zheng Huangtao H Zhu Rongfeng R Han Yu Y Yang Mingdong M Li Kai K Ji Xiong X Chen Peng R PR
Cell 20230301 5
A generalizable strategy with programmable site specificity for in situ profiling of histone modifications on unperturbed chromatin remains highly desirable but challenging. We herein developed a single-site-resolved multi-omics (SiTomics) strategy for systematic mapping of dynamic modifications and subsequent profiling of chromatinized proteome and genome defined by specific chromatin acylations in living cells. By leveraging the genetic code expansion strategy, our SiTomics toolkit revealed di ...[more]
Similar Datasets
Project description:By coupling the mass spectrometry, we developed the Site-Link strategy based on the GCE method for analyzing the interacting proteome of histone lysine acylations in living cells. As K* could mimic the natural lysine to be recognized by natural lysine synthase, histones bearing the site-specifically incorporated K*acyl, should also be also integrated into nucleosomes and further compacted into chromatin similar as Kacyl with cell passaging. The subsequent 365-nm light irradiation could activate the diazirine and covalently capture potential effector proteins on native chromatin in living cells. The effector proteins could be identified after gel-based proteomics and MS/MS analysis of the crosslinked histone-effector complexes.
2023-03-06 | PXD029505 | Pride
Project description:By coupling the mass spectrometry, we developed the Site-Link strategy based on the GCE method for analyzing the interacting proteome of histone lysine acylations in living cells. As K* could mimic the natural lysine to be recognized by natural lysine synthase, histones bearing the site-specifically incorporated K*acyl, should also be also integrated into nucleosomes and further compacted into chromatin similar as Kacyl with cell passaging. The subsequent 365-nm light irradiation could activate the diazirine and covalently capture potential effector proteins on native chromatin in living cells. The effector proteins could be identified after gel-based proteomics and MS/MS analysis of the crosslinked histone-effector complexes.
2023-03-06 | PXD033959 | Pride
Project description:C13, N15 isotope labelled heavy lysine bearing the modification group, such as cr and bhb, were site-sepcifically incorporated into histone H3K56 in living cells for measurement of the modification life of H3K56cr and H3K56bhb without iinterference caused by endogenous histone modification. Utilizaing mass spectrometry, we compared with H3K56cr/bhb peptide with the H3K79 peptide as internal standard at different times. The quantitation analysis of H3K56cr and H3K56bhb at different time points can be comparative.
2023-03-06 | PXD033958 | Pride
Project description:With the evolution of the PylRS system, we identified the PylRS variants for incorporation of all synthesized Kacyl and Kacyl* in the full-length H3 protein both in E. coli and mammalian cells, which covered the tail domain (eg. K23, K27, K56) as well as the globular domain that are difficult for chemical synthesis (eg. K64, K79, K122).
2023-03-06 | PXD029504 | Pride
Project description:Background: Calorie restriction (CR) is the only intervention known to extend lifespan in a wide range of organisms, including mammals. However, the mechanisms by which it regulates mammalian aging remain largely unknown and the involvement of the TOR and Sirtuin pathways (which regulate aging in lower organisms) remain controversial. Femaleness is a second phenotype generally associated with longevity but the relationship between sex-biased and CR-induced gene expression remains undetermined. Methodology/Principal Findings: We generated microarray gene expression data from livers of male mice fed high calorie or CR diets, and we find that CR significantly changes the expression of over 3,000 genes, many between 10- and 50-fold. We compare our data to the GenAge database of known aging-related genes and to prior microarray expression data of genes expressed differently between male and female mice. CR generally feminizes gene expression and many of the most significantly changed individual genes are involved in aging, hormone signaling, and p53-associated regulation of the cell cycle and apoptosis. Among the genes showing the largest and most statistically significant CR-induced expression differences are Ddit4, a key regulator of the TOR pathway, and Nnmt, a regulator of lifespan linked to the Sirtuin pathway. Using Western analyses, we confirmed post-translational inhibition of the TOR pathway. Conclusions: Our data show that CR induces widespread gene expression changes and acts through highly evolutionarily conserved pathways, from microorganisms to mammals, and that its life-extension effects might arise partly from a shift toward a gene expression profile more typical of the longer-lived female sex. Keywords: Two-class gene expression comparison. Calorie restriction (CR) versus HIGH CALORIE feeding. Design of the experiment is a two-class paired design in which the two classes are HIGH CALORIE and CALORIE RESTRICTION (CR) dietary regimens fed to mice, resulting in 15 HIGH CALORIE microarrays and 8 CR microarrays; 23 total microarrays. Four HIGH CALORIE and 2 CR microarrays were produced using pools of 3 RNA samples for each microarray (6 pools of 3, plus 17 individual samples = 35 individual mouse liver samples, 23 microarrays). Total liver RNA was labeled directly. Reference RNA: Stratagene Universal Mouse Reference RNA.
2008-12-30 | E-GEOD-11244 | biostudies-arrayexpress
Project description:Crosslinking mass spectrometry (CXMS) is a powerful technique to study protein structures and interactions. However, software tools for CXMS data analysis suffer from poor speed and credibility, especially at a proteome scale. Here, we introduce pLink 2, a search engine with higher speed and credibility for proteome-scale identification of crosslinked peptides. With a novel two-stage open search strategy facilitated by fragment indexing, pLink 2 is ~40 times faster than pLink 1 and 3~10 times faster than Kojak. Furthermore, four new analysis methods, using simulated datasets, synthetic datasets, 15N metabolically labeled datasets, and entrapment databases, were designed to evaluate the credibility of ten state-of-the-art search engines, this systematic evaluation showed that pLink 2 achieved the highest precision and sensitivity. Finally, four whole-cell lysate datasets, previously analysed by pLink 1 in months, were reanalysed by pLink 2 only in days, with up to 27.4% more crosslinks identified. The new search engine pLink 2 and the new evaluation methods are well positioned to become new benchmarks in the CXMS research field.
2019-07-26 | PXD012109 | Pride
Project description:The identification of genes transcriptionally silenced by DNA hypermethylation is important in understanding the molecular basis of epigenetically regulated biological processes such as X chromosome inactivation, genomic imprinting, and cancer development. Our previously developed methyl-CpG targeted transcriptional activation (MeTA) method reactivates epigenetically silenced genes by using a methyl-CpG binding domain from MBD2 with a transcriptional activation domain. We applied either MeTA or a conventional DNA demethylating agent, 5-aza-cytidine (Aza-CR), to a human embryonic kidney cell line 293T and analyzed gene expression profiles by microarray; 138 and 202 genes that are upregulated 5-fold or more were identified by MeTA and Aza-CR, respectively. The top ten upregulated genes detected by MeTA were further analyzed. We found associations between expressional restorations by MeTA, methylation status, and NFkB(AD)-MBD fusion protein bindings in CpG islands (CGIs) around the transcription start site of the genes. Importantly, MeTA can upregulate genes meeting the stringent criteria of CGIs defined by Takai and Jones at the promoter region at higher frequency; 109 of 138 (79.0%) genes in MeTA vs. 121 of 202 (59.9%) genes in Aza-CR. Interestingly, only 27 genes were upregulated by both methods; MeTA may identify methylated genes that show low levels of induction by the DNA demethylating agents; demethylating agents may also induce factors that help re-expression of genes that harbor less stringent or no CGIs. These results suggest that microarray coupled with MeTA (MeTA-array) is an efficient alternative way to identify transcriptionally silenced genes by DNA hypermethylation. 293T cells were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection. In contrast, 293T cells were treated with 5-aza-cytidine (Aza-CR, 25 uM) or the same volume of PBS for 96 h and medium replaced every 24 h.
2011-06-03 | E-GEOD-25981 | biostudies-arrayexpress
Project description:Linker histones are involved in the formation of higher-order chromatin structure. Although linker histones have been implicated in the regulation of specific genes, it still remains unclear what their principal binding determinants are and how their repressive function in vitro can be reconciled with presumed broad binding in vivo. We generated a full genome, high resolution binding map of linker histone H1 in Drosophila Kc cells, using DamID. H1 binds at similar levels across much of the genome, both in classical euchromatin and heterochromatin. Strikingly, there are pronounced dips of low H1 occupancy around transcription start sites of active genes and at many distant cis-regulatory sites. H1 dips are not due to lack of nucleosomes. Rather, all regions with low binding of H1 show enrichment of the histone variant H3.3 which itself has been linked to high nucleosome turnover. Upon knockdown of H3.3, we find that H1 levels increase at sites previously not covered with H1 with a concomitant increase in nucleosome repeat length. These changes are independent of transcriptional changes. Our results show that the H3.3 protein counteracts association of H1 at genomic sites with high rates of histone turnover. This antagonism provides a mechanism to keep diverse genomic sites in an open chromatin conformation. For this study, we generated DamID profiles of histone H1 and RpII18 in Drosophila Kc167 cells. Additionally, we generated H1 profiles in cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Nucleosome positioning profiles were generated in untreated cells and cells treated with RNAi against white, H3.3B, or H3.3A and H3.3B. Profiles of expression changes were generated for H3.3B RNAi and H3.3A and H3.3B RNAi. DamID experiments for H1 and RpII18 were performed in Drosophila cell cultures. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Formaldehyde-assisted isolation of regulatotry elements (FAIRE) was performed in Drosophila Kc167 cells. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing over non-crosslinked genomic DNA. Nucleosome positioning profiles were made by hybridizing mononucleosomal DNA over MNase digested purified genomic DNA on 380k NimbleGen arrays with 10 bp probe spacing. Expression profiles were made as H3.3 RNAi over white RNAi cohybridizations on spotted INDAC long oligo arrays. Every experiment was done in duplicate in the reverse dye orientation.
2010-06-21 | E-GEOD-16883 | biostudies-arrayexpress
Project description:Identification and characterization of epigenetically silenced genes is very important for cancer research. Particularly, information of hypermethylated genes provides clues to understand roles of epigenetics in tumorigeneses, and genes frequently methylated in a tumor-specific manner can be used as tumor markers. DNA methylation inhibitors such as 5-aza-cytidine or 5-aza-2M-bM-^@M-^Y-deoxycytidine were widely used to search epigenetically silenced genes. However, these inhibitors frequently upregulate genes whose promoters remain unmethylated. We tried to improve the specificity and sensitivity in detecting such methylation-mediated silenced genes in cancer and successfully developed a new method termed M-bM-^@M-^\methyl-CpG targeted transcriptional activation (MeTA)M-bM-^@M-^] by using a transcriptional activating fragment with a methyl-CpG binding domain (MBD) that specifically recognizes and binds to methylated DNAs. Because MBD proteins in fact mediate transcriptional repression of tumor suppressor genes associated with promoter hypermethylation in cancer, MeTA is thought to be one of the ideal methods to search such genes. In the present study, we applied this method to three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control). All of these cell lines have already been analyzed their expression profiles by 5-aza-2M-bM-^@M-^Y-deoxycytidine. We first analyzed the expression of five genes by RT-PCR with Southern hybridization, NEFH, NPTX2, SFRP1, TIMP3, and UCHL1; these genes are known to be methylated in at least any one of these cancer cell lines. Upregulation by M-bM-^@M-^\MeTAM-bM-^@M-^] was confirmed in all of these genes. Then we searched for upregulated-genes, by two-folds or more, in all the three cancer cell lines after MeTA; nineteen such upregulated genes were identified. Among these, sixteen genes except NEFH, HOXA9, and CLDN5 have not been reported previously using the conventional DNA methylation inhibitors. Methylation status of two genes, SLC32A1 and CSMD2, were further analyzed by methylation-specific PCR and found that SLC32A1 and CSMD2 were methylated in 100% (21/21) and 83% (15/18) pancreatic cancer cell lines analyzed, respectively. Our results suggest that M-bM-^@M-^\MeTAM-bM-^@M-^] is a highly efficient method to isolate methylation-mediated transcriptionally silenced genes in human pancreatic cancer and that this method can be applied to other types of human cancer. Three representative pancreatic cancer cell lines, AsPC-1, MIA PaCa-2, and PANC-1, with a normal pancreatic ductal epithelial cell line HPDE (as the control) were transfected with pcDNA6/myc-His vector or pcDNA6-3xFLAG-NFkB (AD)-MBD and were harvested 48 h after transfection.
2012-12-11 | E-GEOD-26215 | biostudies-arrayexpress
Project description:The local protein composition of chromatin is important for the regulation of transcription and other functions. By integrative analysis of genome-wide binding maps of 53 broadly selected chromatin components in Drosophila cells, we show that the genome is segmented into five principal chromatin types that are defined by unique, yet overlapping combinations of proteins, and form domains that can extend over >100 kb. We identify a novel repressive chromatin type that covers about half of the genome and lacks classic heterochromatin markers. Furthermore, transcriptionally active euchromatin consists of two distinct types that differ in molecular organization and H3K36 methylation, and regulate distinct classes of genes. Finally, we provide evidence that the different chromatin types act as guides that help to target DNA-binding factors to specific subsets of their recognition motifs. These results uncover basic principles of chromatin organization in a higher eukaryote. For this study, we generated whole-genome DamID binding profiles of 45 chromatin proteins in Drosophila Kc167 cells. Additionally, we perused published binding data of 8 chromatin proteins and generated a binding profile of one exogenous (yeast) DNA binding factor in Kc167 cells. On the same array platform, we obtained ChIP-on-chip profiles of histone H3, H1, H3K9me2, H3K27me3, H3K4me2, and H3K79me3. See supplementary files below. Gene expression was measured by RNA tag profiling. See GeneCounts supplementary file below. [1] RNA tag sequences were optained on an Illumina GAII with the digital gene expression (DGE) module from duplicate RNA samples. [2] All DamID and ChIP experiments were done in Drosophila Kc167 cells in duplicate. Samples were hybridized to 380k NimbleGen arrays with 300 bp probe spacing. Every experiment was done in duplicate in the reverse dye orientation, where Dam-fusion material was hybridized over Dam-only material. For ChIP, immunoprecipitated material was hybridized over ChIP input material. 18 previously-submitted Samples were included in this study. 10 of 18 Samples have been renormalized for the GSE22069 study: GSM509087, GSM509088, GSM509089, GSM509090, GSM509091, GSM509092, GSM509093, GSM509094, GSM509095, GSM509096 New GSM accession numbers have been issued for these 10 samples. 8 of 18 Samples are identical in the original studies and in GSE22069: GSM423290, GSM423291, GSM423298, GSM423299, GSM493592, GSM493593, GSM509085, GSM509086 [3] The genomic locations in files GSE22069_norm_aggregated_discretized_tiling_arrays.txt and GSE22069_norm_aggregated_tiling_arrays.txt are relative to FlyBase release 5 (BDGP R5/dm3).
2010-10-01 | E-GEOD-22069 | biostudies-arrayexpress